The epsilon-globin gene is autonomously regulated and is not significantly expressed in adult erythroid cells. We use continuous cell lines, transgenic mice and primary cultures of human adult erythroid cells to examine the silencing of the epsilon globin gene and the role of the epsilon globin silencer located in the 5' region of the epsilon globin gene. We have previously identified YY1 and GATA-1 binding motifs within the epsilon-globin silencer and demonstrated that mutation of these sites is able to relieve the negative regulatory effect of the epsilon-globin silencer in transient transfection assays. We have now examined the effect of these mutations on transcription activity of the epsilon globin gene in transgenic mice and in primary adult human erythroid cultures. In transgenic mice, a construct containing the epsilon-globin gene and extending 5' to include HS2, mutation of the GATA-1 and YY1 sites within the epsilon globin silencer is able to relieve suppression of epsilon globin gene expression. When transfected into primary adult human erythroid cultures, these mutations also result in activation of the epsilon globin promoter. When combined with a heterologous promoter, the epsilon-globin silencer has little suppressor activity in K562 cells, but is able to reduce transcription in the adult erythroid cultures indicating that the silencer does not require the LCR or a globin promoter for suppressor activity. These data suggest that the human adult erythroid cultures are a useful alternative to the transgenic mouse in studying globin gene expression and that the epsilon-globin silencer is active even in the absence of the LCR or epsilon-globin promoter. To examine the role of post-transcriptional processing on globin gene expression, we studied globin RNA transcripts in human adult erythroid cells which express primarily adult globin genes. Although not expressed, epsilon globin transcripts are detected, with exon I levels comparable to that of gamma-globin exon I and much higher than exons II and III. All or most of the globin transcripts for beta-globin found in the cytoplasm or nucleus are correctly processed while epsilon globin transcripts that detected both in the cytoplasm and nucleus of cells without expression of the corresponding protein are largely unspliced (containing one or two intervening sequences). Hemin, which increases gamma-globin expression and promotes correct splicing. These studies suggest that in addition to changes in transcription rates, changes in completion or processing of globin RNA transcripts may contribute to the developmental regulation of the hemoglobin phenotype and its modulation by pharmacological agents.